This invention relates to spectrometry instrumentation in general and in particular examples to fluorescence, phosphorescence and luminescence spectrophotometry.
A fluorescence spectrophotometer usually comprises a flash light source, an excitation monochromator or filter, a sample cell containing a sample to be analysed, an emission monochromator or filter, a photodetector and signal processing electronics. A specific wavelength of light from the flash source, as selected by the excitation monochromator or filter, is directed into the sample cell and resultant fluorescence light from the sample enters the emission monochromator or filter. A specific wavelength of the fluorescence light, as selected by the emission monochromatolr or filter, is directed onto the photodetector to produce an electrical signal corresponding to the intensity of the fluorescent light. Such an instrument may be arranged to make a fluorescence, phosphorescence or luminescence measurement. Fluorescence measurements relate to light which is emitted virtually immediately by a sample upon its exposure to the excitation light, whereas phosphorescence measurements relate to the light emitted from the sample a short characteristic time after its exposure to the excitation light. Luminescence measurements are taken by measuring the emitted light from a sample without exposing the sample to excitation light. Such measurements are used to characterise substances, with fluorescence measurements in particular having wide application in the biotechnical field for characterising DNA and other proteins, for example using fluorofors.
It is known in spectrometry instruments in general, and in spectrophotometers for fluorescence, phosphorescence and luminescence measurements, to provide exchangeable accessories. Generally these may provide different sample presentation facilities, for example a liquid sample presentation accessory may be exchanged for one which provides for presentation of a solid state sample. Different accessories may also provide for temperature control of samples via Peltier, Dewar or other cryostat devices, successive feeding of multiple samples to a reading location, or multiple sample carriers such as a well plate and reader therefor.
In order not to compromise test results, it is important that the exchangeable accessories for a spectrometer be repeatably and accurately locatable on the instrument. Prior art arrangements for doing this, which involve screw threaded attachment of one part to another, generally do not facilitate rapid exchange of one accessory for another.
As described above, the capability to make phosphorescence measurements (that is, phosphorescence emission intensity versus time) is included in some fluorescence spectrophotometers. To collect phosphorescence intensity versus time data that results from a short pulse of excitation light, it is necessary to repetitively measure the emission intensity at a time short enough to adequately define the relationship. The capturing of a data point can be done relatively quickly via a sample and hold circuit, however the measurement and digitisation of that data point typically takes a reasonable length of time. Such data conversion often takes longer than the required interval between successive measured points. By way of example, adequate definition of the emission time relationship may require measurement of the emission intensity at 1 microsecond intervals yet the digitisation of a single emission datum may take, say, 19.5 microseconds. For this reason, the prior art technique is to use a sampling approach. In this arrangement, the excitation light pulse is generated repetitively at a constant interval. The interval must be long enough for the emission from one pulse to have fallen substantially to zero before the next pulse is applied. After each excitation pulse a single emission intensity is measured at a controlled time after the excitation pulse so as to give a single datum of the emission time relationship. For each successive cycle the time interval between the excitation and capturing of emission intensity is modified so as to build up a complete picture of the overall emission versus time relationship. In the example given for the first cycle the time delay could be 1 microsecond. For the second cycle the time delay may be 2 microseconds. For the third the delay will be 3 microseconds and so on.
The problem with this approach is that the interval between excitation pulses must be long enough to allow the emission to die away substantially to zero between one pulse and the next. At the same time many cycles are needed to build up a comprehensive picture of the emission versus time relationship. The overall measurement is thus slow. For example, again referring to the above example of one microsecond intervals between data points, if data covering two milliseconds is desired then 2000 data points will need to be collected. If the time for the emission to substantially fall to zero is 10 milliseconds, it will take 20 seconds to complete the 2000 measurement cycles.
According to a first aspect the present invention provides a spectrometry instrument and an exchangeable accessory therefor including a manually operable mechanism for attaching the exchangeable accessory to the instrument, the mechanism including a manually rotatable camming means associated with one of the accessory or the instrument, a male member associated with the other of the accessory or the instrument. The male member having a camming surface which is engageable by the camming means, wherein the accessory is positionable on the instrument in a predetermined location and the camming means is manually rotatable to engage the camming surface of the male member and thereby lock the accessory on the instrument in the predetermined location.
In spectrometry instruments which have exchangeable accessories, it would be advantageous if the instrument could detect if an accessory has been attached and if so, to identify what accessory it is. The advantages of this include the instrument""s set up and programming for use with a particular accessory being able to be automatically established. Also for those accessories that include electrical componentry, such as stepper motors, it would be advantageous to detect the presence of such a component.
According to a second aspect the present invention provides a spectrometry instrument including an electrical circuit for identifying anyone of a plurality of exchangeable accessories which are connectable to the instrument, the electrical circuit including a voltage source and means for generating an identifying voltage therefrom, wherein each accessory includes at least one circuit element such that connection of an accessory to the instrument alters the identifying voltage to a value which is uniquely dependent upon the accessory which is connected to the instrument.
The accessory recognition circuitry may be such that it recognises the presence of an electric motor of an accessory. In this case a voltage divider can be arranged to provide a logic high signal in the presence of a motor by virtue of the motor winding completing a circuit between the voltage source and the voltage divider. In the absence of the motor, the circuit is open and a logic low signal is derived from the voltage divider.
Preferably the spectrometer includes circuitry for identifying an accessory and further circuitry for determining the presence or absence of an electric motor in that accessory.
For a spectrometer with a capacity to have a number of different accessories connected thereto at the same time, each connection socket for each accessory may include accessory recognition circuitry as above described. In this arrangement, the signal line for the identifying voltage from each circuit may be connected to a multiplexer for input to a microprocessor of a computer.
In a third aspect the present invention provides a method and apparatus for reducing the time for measuring a number of data points for determining a phosphorescence decay characteristic (that is, phosphorescence emission intensity versus time) of a sample.
According to this third aspect, there is provided a method of determining a phosphorescence decay characteristic of a sample or at least a portion thereof, including
i) exposing the sample to a first excitation hash of light,
ii) measuring the intensity of a decaying phosphorescence light signal from the sample caused by the first excitation flash at each of a sequence of measurement points which commence a controlled time after the first excitation flash and are separated by controlled times,
iii) exposing the sample to a second excitation flash of light and
iv) measuring the decaying phosphorescence light signal from the sample caused by the second excitation flash at each of a sequence of measurement points which commence a controlled time after the second excitation flash and are separated by controlled times, wherein the time instants to the first and subsequent measurement points from the second excitation flash lie between the first and subsequent measurement points respectively from the first excitation flash,
v) assembling the phosphorescence measurements into time sequence to produce a phosphorescence decay (characteristic, or a portion thereof, for the sample.
The assembly of the phosphorescence measurements into time sequence results in the measured data points from the second excitation flash being interleaved with those from the first excitation flash.
In some cases as the phosphorescence emission from a sample decays, the time interval between the data points which is required to adequately define the phosphorescence characteristic becomes longer. The above described method, in relating to determining possibly only a portion of a phosphorescence decay characteristic, recognises that after a certain time, the necessary time interval between data points to adequately define the characteristic may be so long as to be able to be sequentially measured from the emission caused by one of the excitation flashes and not both. Thus the above described method may be applied only for determining an initial or any particular predetermined portion of a decay characteristic.
The time intervals in step (ii) established by the controlled times are greater than the measurement and digitisation time. These intervals may be controlled in the sense they are prior determined or computed during the data collection process (that is, they are computed xe2x80x9con the flyxe2x80x9d from the time for measurement and digitisation of data). The time intervals between measured data points may be uniform or vary from one interval to the next. Similarly, the time intervals in step (iv) established by the controlled times may be prior determined or determined by computation during the data collection process.
The method may be extended wherein further excitation flashes are initiated and further phosphorescence emission intensity measurements taken which result from each such further excitation flash, the further phosphorescence measurements for each such further excitation flash being taken at controlled times (ie., prior determined or computed times as above described) such that each such further phosphorescence measurement can be interleaved between phosphorescence measurements resulting from earlier excitation flashes. That is, steps (iii) and {iv) may be repeated as often as necessary until all required measured points are obtained.
According to this third aspect of the invention there is also provided apparatus for performing the above described method. This apparatus comprises a spectrophotometer and means for controlling the spectrophotometer, said means for controlling being such as to acquire sequential phosphorescence emission measurements data from each of a number of excitation cycles applied to a sample in the spectrophotometer and to assemble that data into a correct time sequence to define a phosphorescence decay characteristic, or a portion thereof, for the sample.
The means for controlling the spectrophotometer may be a suitably programmed computer or a dedicated device or circuitry.
Preferably this apparatus includes a manually operable mechanism for attaching an exchangeable accessory as described herein above. The apparatus also preferably includes an accessory recognition circuit as also described hereinabove.
The following detailed description with reference to drawings is provided to give a better understanding of the invention and to show how it may be carried into effect in all its aspects. This description and the drawings are given by way of non-limiting example only and are not to be interpreted as limiting the generality of the preceding description.